Protein-coding mutation in Adcy3 increases adiposity and alters emotional behaviors sex-dependently in rats

Abstract

Objective: Adenylate cyclase 3 (Adcy3) has been linked to both obesity and major depressive disorder. We identified a protein-coding variant in the transmembrane (TM) helix of Adcy3 in rats; similar obesity variants have been identified in humans. This study investigates the role of a TM variant in adiposity and behavior.

Methods: We mutated the TM domain of Adcy3 (Adcy3^mut/mut) and created a heterozygous knockout (Adcy3^+/-) in Wistar Kyoto (WKY) rats. Wild-type, Adcy3^+/-, and Adcy3^mut/mut rats were fed a high-fat diet for 12 weeks. We measured body weight, fat mass, glucose tolerance, food intake, metabolism, emotion-like behaviors, memory, and downstream proteins.

Results: Adcy3^+/- and Adcy3^mut/mut rats weighed more than wild-type rats due to increased fat mass. There were key sex differences: adiposity was driven by increased food intake in males but by decreased energy expenditure in females. Adcy3^mut/mut males displayed increased passive coping and decreased memory, whereas Adcy3^mut/mut females displayed increased anxiety-like behavior. Adcy3^mut/mut males had decreased hypothalamic cAMP-response element binding protein (CREB) signaling, with decreased phospho-AMP-activated protein kinase (p-AMPK) signaling in both sexes.

Conclusions: The ADCY3 TM domain plays a role in protein function via p-AMPK and CREB signaling. Adcy3 may contribute to the relationship between obesity and major depressive disorder, and sex influences the relationships between Adcy3, metabolism, and behavior.

FIGURE 1

Development of adenylate cyclase 3 (Adcy3) knockout (KO) and Adcy3^mut/mut rats and confirmation of Adcy3 expression. (A) Adcy3 KO rats have a single base pair deletion in the transmembrane (TM) domain, causing a frameshift mutation and whole-body KO of the gene. Because Adcy3 KO is lethal, Adcy3^+/− rats were used for experiments. Adcy3^mut/mut rats have a three-base pair deletion in the same TM region, causing a F122delV123L protein-coding mutation. (B) Adcy3^+/− males (M) and females (F) express less Adcy3 mRNA than wild-type (WT) rats in retroperitoneal white adipose (RetroFat). Adcy3^mut/mut rats do not have altered Adcy3 mRNA expression in RetroFat. (C) M and F Adcy3^+/−rats express less ADCY3 in RetroFat than WT rats. Adcy3^mut/mut rats do not have altered ADCY3 RetroFat expression. (D) Representative Western blot image comparing ADCY3 expression across genotypes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Mean ± SEM. t test, *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 2

Study design, body weight, and body composition in Adcy3^+/− and Adcy3^mut/mut rats. (A) Timeline of the study shown in weeks of age. Metabolic phenotyping included weekly measurement of body weight and cage-wide food intake, echo magnetic resonance imaging (EchoMRI), intraperitoneal glucose tolerance test (IPGTT), individual housing to measure food intake, TSE PhenoMaster chambers, and euthanasia (sac). Behavioral phenotyping included the open field test (OFT), novel object recognition test (NOR), and forced swim test (FST). (B) Adcy3^+/− males (M) and females (F) gain more weight than wild-type (WT) rats over the course of the study. (C) Adcy3^mut/mut M and F gain more weight than WT rats over the course of the study. (D) There were no differences in fat mass prior to high-fat diet (HFD) start, except in Adcy3^mut/mut M, which have slightly more fat mass than WT M. (E) After 8 weeks on diet (WOD), all four groups have more fat mass than WT rats. Mean ± SEM. t test or repeated-measures ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 3

Adcy3^+/− and Adcy3^mut/mut fat pads and adipocytes at euthanasia (sac). (A) Adcy3^+/− males (M) and females (F) have larger retroperitoneal (RetroFat), gonadal, and omental fat (OmenFat) pads at sac than wild-type (WT) rats. (B) Adcy3^mut/mut M and F also have larger RetroFat, gonadal fat, and OmenFat pads at sac than WT rats. (C) Adcy3^+/− and Adcy3^mut/mut M and F all have significantly larger adipocytes than WT rats. (D) Representative images of hematoxylin & eosin-stained RetroFat from female rats. Scale bars = 250 μm. (E) Relative frequency distributions of adipocyte size for each group. Mean ± SEM. t test, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 4

Sex differences in the cause of adiposity in Adcy3^+/− and Adcy3^mut/mut rats as measured in TSE PhenoMaster chambers. Plots of 24 h average hourly energy expenditure (EE), cumulative EE, respiratory exchange ratio (RER), and cumulative food intake in (A) Adcy3^+/− males (M), (B) Adcy3^+/− females (F), (C) Adcy3^mut/mut M, and (D) Adcy3^mut/mut F compared with wild-type (WT) rats. Adcy3^+/− and Adcy3^mut/mut M have increased food intake, whereas Adcy3^+/− and Adcy3^mut/mut F have decreased EE. Only Adcy3^+/− M had increased RER. Gray shading indicates dark cycle. Mean ± SEM. ANCOVA, *p < 0.05 and **p < 0.01.

FIGURE 5

Cage-wide and individual food intake in Adcy3^+/− and Adcy3^mut/mut rats. (A) No differences in cage-wide weekly food intake in Adcy3^+/− males (M) or (B) Adcy3^+/− females (F). (C) No differences in individual food intake over 1 week in Adcy3^+/− M or F. (D) Adcy3^mut/mut M consume more food than wild-type (WT) M. (E) No differences in cage-wide weekly food intake in Adcy3^mut/mut (F) Adcy3^mut/mut M, but not F, consume more food than WT rats when measured individually over 1 week. Mean ± SEM. t test or repeated-measures ANOVA, *p < 0.05. HFD, high-fat diet.

FIGURE 6

Measures of glucose tolerance and insulin resistance in Adcy3^+/− and Adcy3^{mut /mut} rats. (A) Only Adcy3^+/− males (M) have increased blood glucose compared with wild-type (WT) rats after an overnight fast. (B) Adcy3^+/− and Adcy3 ^mut/mut M have increased fasting insulin after an overnight fast, with no significant differences in females (F). (C) Adcy3^+/− and Adcy3^mut/mut M have increased insulin resistance compared with WT rats, as measured by homeostatic model assessment of insulin resistance (HOMA-IR). There were no significant differences in HOMA-IR in F. Mean ± SEM. t test, *p < 0.05 and **p < 0.01.

FIGURE 7

Adcy3^mut/mut behaviors in the open field test (OFT), novel object recognition test (NOR), and forced swim test (FST). (A) Adcy3^mut/mut females (F), but not males (M), spend significantly less time in the center than wild-type (WT) rats. (B) Adcy3^mut/mut M rear significantly more and Adcy3^mut/mut F rear significantly less than WT rats. (C) Adcy3^mut/mut M tend to groom more than WT M. (D) There were no differences in line crossings in Adcy3^mut/mut M or F. (E) WT M, WT F, and Adcy3^mut/mut F all spend significantly more time with the novel object than the familiar object. Adcy3 ^mut/mut M do not spend significantly more time with the novel object than the familiar object, suggesting impaired memory.(F) Adcy3^mut/mut M tend to spend less time with the novel object relative to WT M. There were no differences in time with the novel object between WT F and Adcy3^mut/mut F. (G) Adcy3^mut/mut M spend significantly more time immobile in the FST than WT M. There were no differences in FST behavior in Adcy3^mut/mut F. Mean ± SEM. t test, *p < 0.05 and ***p < 0.001.

FIGURE 8

Molecular analyses of downstream proteins in the hypothalamus in Adcy3^+/− and Adcy3^mut/mut rats. (A) Adcy3^mut/mut males (M) have significantly less protein kinase A (PKA) than wild-type (WT) M. Adcy3^mut/mut females (F) and Adcy3^+/− M and F do not have altered PKA levels. (B) Representative Western blot images of PKA. (C) Adcy3^mut/mut F have significantly less phospho-AMP-activated protein kinase (p-AMPK)/total AMPK compared with WT rats. Adcy3^mut/mut M tend to have less p-AMPK/total AMPK compared with WT rats. Adcy3^+/− M and F do not have altered p-AMPK/total AMPK. (D) No group has changes in total AMPK compared with WT rats. (E) Representative Western blot images of p-AMPK and total AMPK. (F) No group has changes in phospho-cAMP-response element binding protein (p-CREB)/total CREB compared with WT rats. (G) Adcy3^mut/mut M have significantly less total CREB compared with WT rats. Adcy3^mut/mut F and Adcy3^+/− M and F do not have altered total CREB. (H) Representative Western blot images of p-CREB and total CREB. α-tubulin was used as a loading control. Mean ± SEM. t test, *p < 0.05 and **p < 0.01.